Use of mannosamine derivatives for the stimulation of neurite growth

ABSTRACT

The present invention relates to the use of peracylated N-acyl-mannosamine derivatives for the preparation of a medicament for stimulating neurite growth.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to German Application No.10147382.6, filed Sep. 26, 2001 and German Application No. 10203308.0,filed Jan. 29, 2002, which applications are incorporated herein fully bythis reference.

The present invention relates to the use of N-acyl-mannosaminederivatives, especially N-propanoyl-1,3,4,6-O-tetraacetylmannosamine andN-cyclopropyl-carbonyl-1,3,4,6-O-tetracyclopropylcarbonylmannosamine forthe stimulation of neurite growth.

BACKGROUND OF THE INVENTION

Sialic acids are carbohydrates which form a family of about 40 naturallyoccurring derivatives of neuraminic acid and which all except one areN-acylated (Schauer et al., 1995; Varki, 1992). They are distinguishedfrom another by N and/or O substituents. N substituents are primarilyacetyl and glycolyl groups. O substituents are, among others, acetyl,lactoyl, methyl, sulfate and phosphate groups. The most abundantrepresentative of sialic acids is N-acetylneuraminic acid (NeuNAc),which is the precursor of all other sialic acids. Sialic acids are theterminal monosaccharides of complex N-glycans and many O-glycanes ofglycoproteins as well as gangliosides.

By different modifications and binding types, the neuraminic acidscontribute to the structural variety of glycoconjugates and have a greatinfluence on numerous biological processes.

While sialinic acids play an important role in various biologicalprocesses, until recently, nothing was known about the importance oftheir N-acetyl or N-glycolyl side chain in these processes. Thephysiological precursor of all natural sialinic acids isN-acetylmannosamine. By the application of non-physiologicalN-acyl-mannosamines or glucosamines whose side chain is extended by oneor more methylene groups, the corresponding neuraminic acids arebiosynthesized (Grunholz et. al., 1981; Kayser et al. 1992).

A precursor such as N-propanoylmannosamine (ManNProp) is metabolizedinto N-propanoylneuraminates and subsequently, as N-propanoyineuraminicacid (NeuNProp), it is incorporated into the membrane proteins of thecells as well as into the serum glycoproteins (gangliosides) (Kayser etal., 1992).

Cell culture experiments have shown that biological processes such asviral infections and cell proliferation are permanently influenced bythus modified neuraminic acids.

DE 197 38 484 discloses the use of N-propanoylmannosamine,N-isopropanoyl-mannosamine and/or N-cyclopropanoylmannosamine (i.e.,more correctly, N-cyclopropylcarbonylmannosamine) for the preparation ofa medicament for myelinization and remyelinization. The capability ofthe above mentioned mannosamine derivatives of effecting myelinizationand remyelinization is based on their property of being stimulators ofoligodendrocytes.

Further, WO 00/07602 describes that particular N-acylmannosamines (suchas N-acetyl-, N-propanoyl-, N-glycolyl-, N-formylmannosamine) aresuitable for the modulation of neuronal growth or for the enhancement orinhibition of neurite growth. However, these compounds have the drawbackthat their effectiveness is rather low. On the other hand, WO 00/07602also discloses O-acylated derivatives of the above mentioned compounds;in particular, N-glycolylmannosamine pentaacetate is mentioned. However,all the glycolylmannosamine derivatives have the drawback of beingantigenic.

Surprisingly, it has been found that special N- and O-acylatedmannosamine compounds can stimulate neurite growth significantly betteras compared to the compounds of WO 00/07602. N-propanoyl- andN-cyclopropylcarbonyltetra-acetylmannosamine have proven particularlysuitable (among the non-O-acylated compounds N-propanoylmannosamine hasbeen found to be most effective.

SUMMARY OF THE INVENTION

Therefore, the present invention relates to

-   (1) the use of N-acylmannosamine derivatives of formula (I)

wherein

-   R is an acyl residue, especially an acetyl (—CO—CH₃), propanoyl    (—CO—C₂H₅), butanoyl (—CO—C₃H₇), pentanoyl (—CO—C₄H₉), hexanoyl    (—CO—C₅H₁₁), cyclopropyl-carbonyl (—CO—C₃H₅), crotonoyl    (—CO—CH═CH—CH₃), levulinoyl (—CO—CH₂—CH₂—CO—CH₃) or azidoacetyl    residue (—CO—CH₂—N₃); and-   each R¹ independently represents an acyl residue, especially an    acetyl residue; for the preparation of a medicament for stimulating    neurite growth;-   (2) a compound of formula (I), wherein R is a cyclopropylcarbonyl    residue, and R¹ represent acyl residues;-   (3) a pharmaceutical composition comprising the compound defined    in (2) above;-   (4) a process for the preparation of the compound defined in (2)    above, comprising the reacting of mannosamine with an activated    cyclopropylcarbonyl compound; and-   (5) a method for stimulating neurite growth in mammals, especially    humans, comprising administration of an acylmannosamine derivative    as defined in (1) to said mammals (preferably to humans).

BRIEF DESCRIPTION OF THE FIGURES

The invention is further illustrated by the following FIGS. 1 to 3:

FIG. 1: On collagen I, PC12 cells were incubated for two days with 100ng of NGF, followed by fixation and staining.

FIG. 2: On collagen I, PC12 cells were incubated for two days with 100ng of NGF and 5 mM N-propanoylmannosamine, followed by fixation andstaining.

FIG. 3 shows the influence of the addition of N-propanoylmannosamine onneurite formation.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, the term “mannosamine derivatives”comprises all derivatives of mannosamine familiar to the skilled personwhich are derived from this compound and which contain “mannosamine” intheir names. The mannosamine derivatives comprise both D and L forms ofmannosamine, and a mixture of the D and L forms. Also included are thepure α and β anomers of the mannosamine derivatives and mixturesthereof.

In the mannosamine compound of formula (1), the acyl residue in thedefinition of R¹ may be any acyl residue familiar to the skilled person.However, it is preferred that R¹, like R, is an acetyl, propanoyl(—CO—C₂H₅), butanoyl (—CO—C₃H₇), pentanoyl (—CO—C₄H₉), hexanoyl(—CO—C₅H₁₁), cyclopropylcarbonyl (—CO—C₃H₅), crotonoyl (—CO—CH═CH—CH₃),levulinoyl (—CO—CH₂—CH₂—CO—CH₃) or azido-acetyl residue (—CO—CH₂—N₃),wherein R is more preferably a propanoyl, isopropanoyl, butanoyl orcyclopropylcarbonyl residue, and R¹ are acetyl residues.

According to the present invention, the residues comprise butanoyl(—CO—C₃H₇), pentanoyl (—CO—C₄H₉) and hexanoyl (—CO—C₅H₁₁), and inaddition to the unbranched residues also their constitutional isomersknown to the skilled person, such as isobutanoyl (—CO—CH—(CH₃)₂),isopentanoyl (—CO—CH₂—CH(CH₃)₂), 2-methylbutanoyl (—CO—CH(CH₃)—C₂H₅)etc.

Further, according to the present invention, the term “crotonoyl”(—CO—CH═CH—CH₃) comprises the two possible geometric isomers, i.e., boththe E and the Z forms, which are known to the skilled person.

In a special embodiment of (1),1-N-propanoyl-3,4,6-O-tetraacetylmannosamine is employed for thestimulation of neurite growth.

The formulation of the medicament according to the invention forstimulating neurite growth according to (1) or the formulation of thepharmaceutical composition (3) may be effected by methods known to theskilled person.

In addition to the compound of formula (I), thesemedicaments/compositions may also contain other pharmaceuticallyacceptable compounds which are familiar to the skilled person, such ascarriers, diluents, etc.

The medicaments (1) are suitable for the treatment ofCNS-neurodegenerative diseases, especially Alzheimer's and Parkinson'sdiseases. Similarly, the method (5) is suitable for treating saiddiseases.

The pharmaceutical composition (3) can be employed both for thestimulation of neurite growth and for myelinization and remyelinization(see DE-A-197 38 484). In addition, the compounds (2) may also beemployed for the preparation of recombinant glycoproteins as describedin WO 00/29567.

The preparation process according to embodiment (4) of the inventioncomprises the reaction of the mannosamine with an activatedcyclopropylcarbonyl compound. Then, to preparepentacyclopropylcarbonylmannosamine, purification of the product fromthe reaction mixture of this (first) acylation step is immediatelyeffected. If derivatives are desired in which acyl residues other thancyclopropyl-carbonyl are present, a mild saponification of the reactionmixture is first effected, followed by a second acylation step withsuitable activated acyl derivatives. Suitable activated acyl compoundsand cyclopropylcarbonyl compounds include anhydrides, acid anhydrides,activated esters etc. Suitable solvents for the acylation reactioninclude polar aprotic solvents, such as pyridine, etc. The reactiontemperature for the acylation steps is from 0 to 30° C., and thereaction time is from 30 min to 24 h.

In the method (5) of the present invention, the compound (I) isadministered together with suitable carriers and diluents. Theadministration may be effected via any possible route (intravenously,orally, etc.). The amount of compound (I) to be administered depends, inparticular, on the patient's body weight and on the route ofadministration and is individually determined by the attendingphysician.

The invention will now be illustrated in more detail by the followingExamples, which are, however, not to be construed as to limit theinvention.

EXAMPLES Example 1 Influence of N-propanoylmannosamine (Hereinafter“ManNProp”) on PC12 Cells After Stimulation with the Growth Factor NGF

It is known that stimulation with nerve growth factor (NGF) causesneuronal differentiation in PC12 cells. At an NGF concentration of 250ng/ml and after three days of incubation, the PC12 cells already form anetwork of neurites.

In order to examine whether the addition of ManNProp has an influence onthe neurite formation by PC12 cells, the suitable neurite lengths fordifferent incubation times were first titrated with different NGFconcentrations. At an NGF concentration of 100 ng and an incubation timeof two days, the cells formed neurites whose length were double thediameter of their cell. Under such conditions, the further course of theexperiments was set up.

The cells were plated with a constant number of cells on culturechambers which consisted of four chambers and were coated with collagenI (col.), poly-D-lysine (PDL) or laminin, and incubated under threedifferent sets of conditions. Each variation was represented induplicate.

In the first chamber, the PC12 cells were incubated with 5 mM ManNProp,and in the second chamber, they were incubated with 100 ng of NGF. Inthe third chamber, 100 ng of NGF plus 5 mM ManNProp was added to thecells. The first and second chambers served as a reference control forthe cells under the influence of NGF and ManNProp. For each experiment,three culture chambers were sown in duplicate. After two days ofincubation, the cells were fixed with paraformaldehyde, stained withcrystal violet and washed with water.

For evaluation, various segments were photographed and measuredaccording to a random choice principle. For the evaluation, only thecells stimulated with NGF which served as a control and the cellsstimulated with NGF plus ManNProp were considered.

Each bar represents three experiments performed on three different days.The relative neurite length was established by CAPA (computer-assistedprocess analysis), and the data were integrated by means of Delta Graph.

All experiments shown here were independently performed on differentdays. The values from Table 1 were additionally represented graphicallyin FIG. 3.

TABLE 1 Enhancement of neurite growth in percent relative to the controlPDL Col. Laminin sd PDL sd Col. sd Laminin 0.5 mM 4 3 28 2.7 2.4 4.3   5mM 5 11 70 2.6 3 5.7  25 mM 17 20 59 3.9 2.9 5.2

1. A compound of formula

wherein R is a cyclopropylcarbonyl residue, and each R¹ independentlyrepresents acyl residues.
 2. A pharmaceutical composition comprising thecompound according to claim
 1. 3. A process for the preparation of thecompound according to claim 1, comprising reacting mannosamine with anactivated cyclopropylcarbonyl compound chosen from cyclopropylcarbonylanhydrides, acid anhydrides, and activated esters to form a productwherein R and each R¹ are cyclopropylcarbonyl.
 4. A process for thepreparation of the compound according to claim 1, comprising: a)reacting mannosamine with an activated cyclopropylcarbonyl compoundchosen from cyclopropylcarbonyl anhydrides, acid anhydrides, andactivated esters to form a product; b) purifying the product obtained instep (a); c) saponifying the purified product obtained in step (b); d)reacting the saponified product obtained in step (c) with an activatedacyl compound chosen from activated acyl anhydrides, acid anhydrides,and activated esters, to form a compound wherein R iscyclopropylcarbonyl and R₁ is an acyl unit other thancyclopropylcarbonyl.
 5. A method for stimulating neurite growth,comprising administering to a mammal with A CNS-neurodegenerativedisease the compound according to claim 1, wherein theCNS-neurodegenerative disease is Alzheimer's or Parkinson's disease. 6.A method of treating a mammal with a CNS-neurodegenerative disease,wherein the neurodegenerative disease is Alzheimer's or Parkinson'sdisease, comprising administering the compound according to claim
 1. 7.A method of treating a mammal with a CNS-disease comprisingadministering the compound according to claim
 1. 8. A method accordingto claim 7, wherein the CNS-neurogenerative disease is Alzheimer'sdisease.
 9. A method according to claim 7, wherein theCNS-neurogenerative disease is Parkinson's disease.
 10. A compoundaccording to claim 1, wherein each R¹ is an acyl residue chosen fromacetyl, propanoyl, isopropanoyl, butanoyl, isobutanoyl, pentanoyl,isopentanoyl, hexanoyl, 2-methylbutanoyl, cyclopropylcarbonyl,crotonoyl, levulinoyl, or azidoacetyl.
 11. A compound according to claim10, wherein each R¹ is acetyl.
 12. A compound according to claim 10,wherein each R¹ is cyclopropylcarbonyl.